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J Vis Exp. 2018 May 25;(135). doi: 10.3791/57350.

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms.

Author information

1
Department of Molecular Cell Biology, University of California, Berkeley; Howard Hughes Medical Institute, University of California, Berkeley.
2
Department of Molecular Cell Biology, University of California, Berkeley.
3
Innovative Genomics Institute, University of California, Berkeley; Biomedical Sciences Graduate Program, University of California, San Francisco; Department of Microbiology and Immunology, University of California, San Francisco; Diabetes Center, University of California, San Francisco.
4
Department of Molecular Cell Biology, University of California, Berkeley; Innovative Genomics Institute, University of California, Berkeley.
5
Innovative Genomics Institute, University of California, Berkeley; Department of Microbiology and Immunology, University of California, San Francisco; Diabetes Center, University of California, San Francisco; Chan Zuckerberg Biohub; Department of Medicine, University of California, San Francisco; UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco.
6
Department of Molecular Cell Biology, University of California, Berkeley; Department of Integrative Biology, University of California, Berkeley.
7
Innovative Genomics Institute, University of California, Berkeley; megan.hochstrasser@berkeley.edu.

Abstract

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has quickly become a commonplace amongst researchers pursuing a wide variety of biological questions. Users most often employ the Cas9 protein derived from Streptococcus pyogenes in a complex with an easily reprogrammed guide RNA (gRNA). These components are introduced into cells, and through a base pairing with a complementary region of the double-stranded DNA (dsDNA) genome, the enzyme cleaves both strands to generate a double-strand break (DSB). Subsequent repair leads to either random insertion or deletion events (indels) or the incorporation of experimenter-provided DNA at the site of the break. The use of a purified single-guide RNA and Cas9 protein, preassembled to form an RNP and delivered directly to cells, is a potent approach for achieving highly efficient gene editing. RNP editing particularly enhances the rate of gene insertion, an outcome that is often challenging to achieve. Compared to the delivery via a plasmid, the shorter persistence of the Cas9 RNP within the cell leads to fewer off-target events. Despite its advantages, many casual users of CRISPR gene editing are less familiar with this technique. To lower the barrier to entry, we outline detailed protocols for implementing the RNP strategy in a range of contexts, highlighting its distinct benefits and diverse applications. We cover editing in two types of primary human cells, T cells and hematopoietic stem/progenitor cells (HSPCs). We also show how Cas9 RNP editing enables the facile genetic manipulation of entire organisms, including the classic model roundworm Caenorhabditis elegans and the more recently introduced model crustacean, Parhyale hawaiensis.

PMID:
29889198
PMCID:
PMC6101420
DOI:
10.3791/57350
[Indexed for MEDLINE]
Free PMC Article

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